Supernatants (sups) from alloantigen and Con A-stimulated human leukocytes and mouse spleen cells contain T cell and macrophage-derived factors which augment in vitro antibody synthesis to sheep red cells by T cell-deficient mouse speen cells. Gel filtration of the human allogeneic sups revealed two factors which individually exhibited minimal helper activity but functioned synergistically to restore the antibody response. The first factor (14,500 Daltons) was: 1) early-acting, 2) inseparable from the T cell activating mediator, LAF, by sequential biochemical purification techniques, and 3) adherent cell-derived. The second factor (45-75,000 Daltons): 1) synergized with LAF and 2) did not induce T cell activation. LAF also synergized with a late-acting factor (25-40,000 Daltons) in alloantigen- and Con A-stimulated mouse spleen cell sups. This synergism was dependent upon the presence of a population of nonadherent, radioresistant accessory cells (presumably pre-T cells) obtained from nude mouse spleens. The results support the hypothesis that LAF induces residual pre-T cells in the "B cell population" to provide an initiator helper function to antigen-stimulated B cells and that the late-acting helper factor induces B cells to undergo terminal differentiation into antibody-forming cells. BIBLIOGRAPHIC REFERENCES: Simon, P. L., Farrar, J.J. and Kind, P.D.: The Xenogenic effect III. Induction of cell-mediated cytotoxicity by alloantigen-stimulated thymocytes in the presence of xenogenic reconstitution factor. J. Immunol 118: 1129-1131, 1977. Farrar, J.J., Koopman, W.J. and Fuller-Bonar, J.: Identification and partial purification of two synergistically-acting helper mediators in human mixed leukocyte culture supernatants. J. Immunol. 1977, in press.